Frequency and the Type of Chromosomal Abnormalities in Patients with Primary Amenorrhea in Northeast of Iran

Objective(s): Primary and secondary amenorrhea are different from each other in that the former refers to a physiological failure in the onset of spontaneous menarche during the time when it is expected. whereas the latter involves the cessation of normal menstruation any time prior to menopause. In this study we aimed to investigate chromosomal abnormalities in patients with Primary Amenorrhea in Northeast of Iran by employing GTG banding. Materials and Methods: Chromosomal analysis was carried out on 180 cases that were referred from different clinics in eastern cities of Iran to our laboratory from 2004 to 2009. We implemented the suggested protocol regarding peripheral blood lymphocyte culture for metaphase chromosome preparation as well as conventional analysis for G-banded chromosome. Results: The karyotype results revealed that 75.55% (n=136) had normal chromosome composition and 24.45% (n=44) showed chromosomal abnormalities. Among the patients with abnormal chromosome constituents 86.36% exhibit numerical aberration and 13.63% showed structural abnormalities. The most frequent abnormality detected was X chromosome monosomy, homogeneous (21 cases –11.66%) or mosaic (8 cases – 4.44%). The other 6 cases (3.33%) had X chromosome structural imbalanced abnormalities (homogeneous or in mosaic). Discussion: As expected, this study confirmed previously reported cytogentic abnormalities in patients with amenorrhea. Although there are percentage differences between these studies and also verities in chromosomal abnormalities, they have still demonstrated the importance of cytogenetic investigations in the etiological diagnosis of amenorrhea.


is was carrie
out on 180 cases that were referred from different clinics in eastern cities of Iran to our laboratory from 2004 to 2009.We implemented the suggested protocol regarding peripheral blood lymphocyte culture for metaphase chromosome preparation as well as conventional analysis for Gbanded chromosome.Results:The karyotype results revealed that 75.55% (n=136) had normal chromosome composition and 24.45% (n=44) showed chromosomal abnormalities.Among the patients with abnormal chromosome constituents 86.36% exhibit numerical abe ration and 13.63% showed structural abnormalities.The most frequent abnormality detected was X chromosome monosomy, homogeneous (21 cases -11.66%) or mosaic (8 cases -4.44%).The other 6 cases (3.33%) had X chromosome structural imbalanced abnormalities (homogeneous or in mosaic).Discussion: As expected, this study confirmed previously reported cytogentic abnormalities in patients with amenorrhea.
lthough there are percentage differences between these studies and also verities in chromosomal abnormalities, they have still demonstrated the importance of cytogenetic investigations in the etiological diagnosis of amenorrhea.

Introduction

Amenorrhea is the absence or abnormal ending of the menses (1).
menorrhea is defined as no menstruation by the age of 14 in the absence of growth or progress of secondary sexual characteristics; no menstruation by the age of 16 despite the presence of normal growth and progress with the appearance of secondary sexual characteristics; and in a woman who has bee menstruating, the absence of menstruation for a length of time equivalent to a total of at least 3 of the previous cycle intervals, or 6 mon hs of amenorrhea (2).

It is classified as primary amenorrhea (PA), which is the collapse menses by the age of 16, or secondary amenor-rhea (SA), in which the menses appears at puberty but is subsequently ceased (1).The prevalence of PA in the United States is less than 1% and the occurrence of SA is 5-7%; no evidence indicates that the occurrence of amenorrhea varies according to national origin or ethni

group (3).

PA is prim
rily caused by: pituitary / hypothalamic disorders (27.8 %); gonadal dysfunction (50.4%); and outflow tract abnormalities (21.8 %) (1).As it can be observed, gonadal / ovarian disorders make up half of the total PA cases.This category of etiology often roots from abnormal sex chromosomes (3).

Amenorrhea is a normal featur in pre pubertal, pregnant, and postmenopausal females and it accounts for 20% of infertility patients.The diagnosis disparity of amenorrhea is wide and can vary from endocrine disorders, genetic abnormalities, ps chological, environmental, and structural anomalies.

Thus karyotyping is one of the standard diagnostic procedures for identifying chromosomal abnormalities involved in this disorder (4)(5)(6).

The division of ch omosomal abnormalities reported is greatly variable, from 15.9% to 63.3% for primary amenorrhea (7)(8)(9)(10)(11)(12)(13). We carried out a retrospective study, with the purpose of establishing the frequency and the type of chromosomal abnormalities, in 180 patients with PA who were referred to our genet cs clinic and cytogenetics laboratory in Ghaem Hospital, Mashhad, Iran, 2004-2009.


Materials and Methods

The study subjects included patients with primary amenorrhea referred (n= 180) for chromosomal analysis to the Cytogenetic Laboratory of Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Iran.The cases were referred from different clinics of the Khorasan province (North East of Iran) between 2004 and 2009.

The age group of the subjects ranged from 14 to 33 years with a mean of 21.7±5.1 years.Pedigrees with details were drawn and in depth clinical evaluation and clinical information were obtained from all subjects.

Primary amenorrhea was described as the absence of menstruation and secondary sexual characteristics in phenotypic women aged 14 years or older or aged 16 or older if secondary sexual characteristics were present.

The diagnosis of primary amenorrhea was determined at the patient's first visit and physical examination was performed to distinguish any secondary sexual characteristics or syndrome features.Laboratory examination and clinical information were obtained from hospital records or the referring physician.

All patients provided informed volunta y approval to participate in the study according to the protocol approved by the local Ethics Committee of MUMS.About 1 ml of blood was supplemented with 8 ml of RPMI medium, 2 ml of fetal bovine serum, 0.1μg/ml of PHA (Phytohemaglutinin) and incubated at 37°C with 5% CO 2 .After 66 hour of incubation, ethidium brom

e (1mg/m
) was added followed by the Colchicine (1mg/ml) at the 67th hour and incubated for another 1.5 hour (14).We obtained cells by applying a hypotonic solution of 0.075M KCl at 37°C for 20 minutes.This was followed by fixation and rinsing three times, using Carnoy's fixative (methanol and acetic acid in a ratio of 3:1).The final outcome was then casted on clean slides, already cooled (15).Twenty five metaphase spread were investigated for each case, and when mosaicism was suspected, at least 50 metaphases were inspected.In order to identify the karyotypes, we took shots of the most revealing meta hases.In case of a translocation or any other abnormality, parents and siblings were also investigated.karyotypic reports were based on the International System for Human Cytogenetic Nomenclature recommendations (ISCN, 2009).

The relative frequency of each diagnostic group was addressed, and the percentage of abnormal cases and the division of

he numerica
and structural abnormalities were determined in each group.The frequencies were compared to similar studies using the Z-test for comparison of two frequencies with unequal variance.


Results

The chromosomal analysis and karyotypes for the patients are summarized in table 1.The karyotype results showed 75.55% with normal chromosome composition (n=136) and 24.45% (n=44) demonstrated chromosomal abnormalities (Figure 1).The karyotype was normal in 136 cases (75.55%), with the PA having an etiology other than chromosomal abnormalities (hypothalamic or pituitary disorders, utero-vaginal abnormalities).

We identified a X chromosome homogeneous monosomy, 45, X in 21 cases (11.7%), a X chromosome monosomy mosaicism in 10 cases (5.6%), and other anomalies in 14 cases (7.8%) (Table 1).

In 13 cases (7.2%) we discove ed a mosaic aneuploidy having mostly the 45, X cell line.We found 2 cases (1.2%) with mosaicism with 1 cell line with X monosomy and the second line with an unbalanced structural abnormality of X chromosome: including isochromosomes Xq (1 case-0.56%),and ring X chromosomes (1 case-0.56%)(Table 1 and Figure 1).


Discussion

It is now clear that genetic disorders could have a considerable health and economic impact on affected individuals, families, and their society.As indicated from previous studies genetic causes of amenorrhea account for approximately 45% of cases-which may be a result of, for example, gonadal dysgenesis, chromosomal disorders,

Müllerian
genesis (14).Several studies have previously been carried out to determine the frequency of sex-chromosome abnormalities among patients with primary amenorrhea (15)(16)(17).It has been showen that chromosomal abnormalities are present in 46%-62% of patients with primary amenorrhea including X aneuploidy, male karyotype, or structural X-chromosome abnormalities which could be presented as X isochromosome, isodicentrics, rings, and deleted or inverted X chromosomes (18).

In the present study the high percentage of chromosomal abnormalities (24.44%) detected in our patients with PA suggests their ma